Precise control of protein targeting within the cell has emerged as a highly important theme in both cellular signaling and the regulation of intracellular membrane- and protein trafficking. In recent years it has been established that the subcellular localization of proteins is defined by small domains that bind to lipids or other components within specific cellular membranes. One of the best characterized examples is the pleckstrinhomology (PH) domain, which is the 1lth most common domain-type in the human genome. About 10% of PH domains bind strongly and specifically to phosphoinositides involved in cell signaling, membrane trafficking and cytoskeletal organization, and act to recruit proteins that contain them to the plasma membrane or elsewhere. In the previous project period we focused on understanding phosphoinositide binding by PH domains at a detailed structural level, allowing comparison with phosphoinositide binding byPX, FYVE, and other domains. In this application we propose structural and biophysical studies of recently discovered phosphoinositide recognition events involved in intracellular trafficking. We also focus on the 90% of PH domains that do not bind strongly to phosphoinositides, in order to understand how these function in membrane recruitment and regulation of their host proteins. Our Specific Aims are:1. To compare specific phosphoinositide binding by novel targets for phosphatidylinositol-(4,5)-bisphosphate(Ptdlns(4,5)P2) and the recently-discovered Ptdlns(3,5)P2 with that seen with PH, PX, and FYVE domains, using quantitative binding studies and X-ray crystallography.2. To determine how PH domains that bind only weakly to phosphoinositides nonetheless drive membrane recruitment of their host proteins. In a recent proteome-wide analysis of S. cerevisiae PH domains we found that 25% fall into this category. We will investigate the role of phosphoinositides in membrane recruitment of these PH domains, as well as the possible existence of additional targets. In addition, we will use structural approaches to distinguish between PH domains that bind strongly and weakly to phosphoinositides. These studies will provide the first view of how Ptdlns(3,5)P2, an important lipid in intracellular trafficking, is recognized by its cellular effectors, and will expand our understanding of the poorly characterized majority of PH domains, which are present in many proteins related to disease states.